Nucleotide sequences which code for the metD gene

ABSTRACT

The invention relates to polynucleotides comprising polynucleotide sequences corresponding to the metD gene and parts thereof that encode polypeptide sequences and parts thereof possessing varying degrees of MetD transcription regulator activity, methods for preparation of L-amino acids, and methods of screening and amplifying polynucleotides encoding polypeptide sequences which comprise varying degrees of MetD transcription regulator activity. Further, the invention relates to animal food additives based on fermentation liquor and containing L-methionine, and to the preparation of such additive.

BACKGROUND OF INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to polynucleotides comprisingpolynucleotide sequences corresponding to the metD gene and partsthereof that encode polypeptide sequences and parts thereof possessingvarying degrees of MetD transcription regulator activity, methods forpreparation of L-amino acids, and methods of screening and amplifyingpolynucleotides encoding polypeptide sequences which comprise varyingdegrees of MetD transcription regulator activity. Further, the inventionrelates to animal food additives based on fermentation liquor andcontaining L-methionine, and to the preparation of such additive.

[0003] 2. Discussion of the Background

[0004] L-Amino acids, in particular L-methionine, are used in humanmedicine and in the pharmaceuticals industry, in the foodstuffsindustry, and, very particularly, in animal nutrition.

[0005] It is known that amino acids are prepared by fermentation fromstrains of Coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocess can relate to fermentation measures, such as, stirring andsupply of oxygen, or the composition of the nutrient media, such as, thesugar concentration during the fermentation, or the working up to theproduct form by, for example, ion exchange chromatography, or theintrinsic output properties of the microorganism itself.

[0006] Methods of mutagenesis, selection, and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and which produce amino acids are obtained in thismanner.

[0007] Methods of the recombinant DNA technique have also been employedfor some years for creating Coryneform bacterium strains, which produceL-amino acid by amplifying individual amino acid biosynthesis genes andinvestigating the effect on the amino acid production. During the timepreceding the present invention, however, it was not known thatattenuated expression of the metD gene would improve L-amino acidproduction yields.

SUMMARY OF THE INVENTION

[0008] An object of the present invention is to provide novel measuresfor improved preparation of L-amino acids or amino acids where theseamino acids include L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan and L-arginine, including their salts (such as methioninehydrochloride or methionine sulfate).

[0009] Another object of the present invention is a novel process forimproving fermentative preparation of the L-amino acids, L-Methionine inparticular. This process includes enhanced bacteria, preferably fromCoryneform bacteria, which express attenuated amounts of MetDtranscription regulator, which is encoded by the metD gene.

[0010] Another object of the present invention is to provide afermentation broth comprising an L-methionine-producing microorganism.

[0011] Another object of the present invention is to provide an animalfood additive, which may be made by a process comprising the steps of:

[0012] a) culturing an L-methionine-producing microorganism in afermentation medium;

[0013] b) concentrating the L-methionine-containing fermentation broth;

[0014] c) removing an amount of from 0 to 100 wt. % of the biomassformed during the fermentation; and

[0015] d) drying of the fermentation broth obtained according to a)and/or b) to obtain the animal feedstuffs additive in the desired powderor granule form.

[0016] Another object of the present invention is to provide a method ofpreparing an animal food additive comprising further steps of:

[0017] e) adding at least one organic substances, including L-methionineand/or D-methionine and/or the racemic mixture D,L-methionine, to theproducts obtained according to a), b) and/or c);

[0018] f) adding auxiliary substances chosen from the group consistingof silicas, silicates, stearates, grits and bran to the substancesobtained according to a) to d) for stabilization and to increase thestorability; or

[0019] g) converting the substances obtained according to a) to e) intoa form stable to the animal stomach, in particular rumen, by coatingwith film-forming agents.

[0020] Another object of the present invention is to provide such abacterium, preferably from Coryneform bacteria, which expressesattenuated metD gene products.

[0021] Another object of the present invention is to provide such abacterium, preferably from Conyneform bacteria, which expressesattenuated MetD transcription regulator activity.

[0022] Another object of the present invention is to provide apolynucleotide sequence encoding a polypeptide sequence with MetDtranscription regulator activity. One embodiment of such a sequence isthe polynucleotide sequence of SEQ ID NO. 1.

[0023] Another object of the present invention is a method of makingMetD transcription regulator or a polypeptide having MetD transcriptionregulator activity. One embodiment of such a sequence is the polypeptidesequence of SEQ ID NO. 2.

[0024] Other objects of the present invention include methods ofdetecting polynucleotides that are homologous to SEQ ID NO: 1 or thosepolynucleotides encoding polypeptides that have having MetDtranscription regulator activity, methods of making such polynucleotidesencoding such polypeptides, and methods of making such polypeptides.

[0025] The above descriptions highlight certain aspects and embodimentsof the present invention. Additional objects, aspects, and embodimentsof the present invention follow in the detailed description of thepresent invention considered together with the Figures.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1: Map of the plasmid pK18mobsacBmetD del

DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless specifically defined, all technical and scientific termsused herein have the same meaning as commonly understood by a skilledartisan of molecular biology.

[0028] “Isolated” refers to a material, i.e. a polynucleotide separatedout of its natural environment.

[0029] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0030] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example, by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures. By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

[0031] “Polypeptides” are understood as meaning peptides or proteins,which comprise two or more amino acids, bonded via peptide bonds.

[0032] The term “enhancement” in this connection describes an increasein the intracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example, byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures. By enhancement measures, in particular over-expression, theactivity or concentration of the corresponding protein is in generalincreased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, up to a maximum of 1000% or 2000%, based on that of thewild-type protein or the activity or concentration of the protein in thestarting microorganism.

[0033] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting.

[0034] Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basicscientific techniques, encompassed by the present invention. See, forexample, Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989) and various references cited therein.

[0035] The invention provides an isolated polynucleotide from Coryneformbacteria, comprising a polynucleotide sequence, which codes for the metDgene, chosen from the group consisting of

[0036] (a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide, which comprisesthe amino acid sequence of SEQ ID NO. 2,

[0037] (b) polynucleotide which codes for a polypeptide, which comprisesan amino acid sequence, which is identical to the extent of at least 70%to the amino acid sequence of SEQ ID NO. 2,

[0038] (c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0039] (d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c),

[0040] the polypeptide preferably has the activity of MetD transcriptionregulator.

[0041] “Transcriptional regulators” are defined herein as proteins whichare able to increase or decrease the transcription level of specificgenes by binding at certain DNA regions.

[0042] It has been found that some transcriptional regulators have aspecific structure called helix-turn-helix-motif. The invention providesthe function of the transcriptional regulator metD as the repression ofgenes which are involved in L-amino acid biosyntheses in particular thebiosynthesis of L-methionine. The attenuation of the transcriptionalregulator metD improves the production of L-methionine in Coryneformbacteria.

[0043] The invention also provides the above-mentioned polynucleotide,this preferably being a DNA which is capable of replication, comprising:

[0044] (i) the nucleotide sequence, shown in SEQ ID NO.1, or

[0045] (ii) at least one sequence which corresponds to sequence (i)within the range of the degeneration of the genetic code, or

[0046] (iii) at least one sequence which hybridizes with the sequencescomplementary to sequences (i) or (ii), and optionally

[0047] (iv) sense mutations of neutral function in (i).

[0048] The invention also provides at least one polynucleotides chosenfrom the group consisting of:

[0049] a) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions 1and 313,

[0050] b) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions314 and 1024,

[0051] c) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions1025 and 1322.

[0052] Further, the invention also provides:

[0053] (a) a polynucleotide, in particular DNA, which is capable ofreplication and comprises the nucleotide sequence as shown in SEQ IDNO.1;

[0054] (b) a polynucleotide, which codes for a polypeptide which,comprises the amino acid sequence as shown in SEQ ID NO. 2;

[0055] (c) a vector containing parts of the polynucleotide according tothe invention, but at least 15 successive nucleotides of the sequenceclaimed,

[0056] (d) Coryneform bacteria in which the metD gene is attenuated, inparticular by an insertion or deletion.

[0057] The invention also provides polynucleotides with a polynucleotidesequence which comprises the complete metD gene or parts thereof,obtainable by screening by means of hybridization of a correspondinggene library of a Coryneform bacterium with a probe which comprises thesequence of the polynucleotide according to SEQ ID NO.1 or a fragmentthereof, and isolation of the polynucleotide sequence mentioned.

[0058] The present invention provides polynucleotides which comprise thesequences according to the invention are suitable as hybridizationprobes for RNA, cDNA and DNA, in order to isolate, in the full length,nucleic acids or polynucleotides or genes which code for MetDtranscription regulator or to isolate those nucleic acids orpolynucleotides or genes which have a high similarity with the sequenceof the metD gene. They are also suitable for incorporation intoso-called “arrays”, “micro arrays” or “DNA chips” in order to detect andto determine the corresponding polynucleotides.

[0059] Polynucleotides, which comprise the sequences according to theinvention, are furthermore suitable as primers, which code for MetDtranscription regulator can be prepared by the polymerase chain reaction(PCR).

[0060] Such oligonucleotides which serve as probes or primers compriseat least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or24, very particularly preferably at least 15, 16, 17, 18 or 19successive nucleotides. Oligonucleotides which have a length of at least31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides are also suitable. Oligonucleotideswith a length of at least 100, 150, 200, 250 or 300 nucleotides areoptionally also suitable.

[0061] The polynucleotides according to the invention include apolynucleotide according to SEQ ID NO. 1 or a fragment preparedtherefrom and also those which are at least 70% to 80%, preferably atleast 81% to 85%, particularly preferably at least 86% to 90%, and veryparticularly preferably at least 91%, 93%, 95%, 97% or 99% identical tothe polynucleotide according to SEQ ID NO. 1 or a fragment preparedtherefrom.

[0062] The polypeptides according to the invention include a polypeptideaccording to SEQ ID NO. 2, in particular those with the biologicalactivity of MetD transcription regulator, and also those which are atleast 70% to 80%, preferably at least 81% to 85%, particularlypreferably at least 86% to 90%, and very particularly preferably atleast 91%, 93%, 95%, 97% or 99% identical to the polypeptide accordingto SEQ ID NO. 2 and have the activity mentioned.

[0063] The invention furthermore relates to a process for thefermentative preparation of amino acids chosen from the group consistingof L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine,L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan andL-arginine using Coryneform bacteria which, in particular, alreadyproduce amino acids and in which the nucleotide sequences which code forthe metD gene are attenuated, in particular eliminated or expressed at alow level.

[0064] The increase of the protein concentration can be analyzed by 1-and 2-dimensional protein gel electrophoresis followed by opticalidentification of the protein concentration while using specificcomputer software. A common method to prepare protein gels usingCoryneform bacteria and to identify the proteins is described by Hermannet al. (Electrophoresis, 22:1712-23 (2001)).

[0065] The concentration of the protein can be also analyzed by Westernblot hybridization techniques while using specific antibodies (Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and subsequentoptical evaluation with computer software commonly used for analyzing ofprotein concentrations (Lohaus und Meyer (1998) Biospektrum 5:32-39;Lottspeich (1999) Angewandte Chemie 111:2630-2647). The activity of DNAbinding proteins can be measured by DNA band shift assays (also known asgel retardation (Wilson et al. (2001) Journal of Bacteriology183:2151-2155). The influence of DNA binding proteins on gene expressioncan be identified by well described reporter gene assays (Sambrook etal., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

[0066] The microorganisms to which the present invention relates canprepare amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of Coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

[0067] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum (C. glutamicum), are inparticular the known wild-type strains

[0068]Corynebacterium glutamicum ATCC13032

[0069]Corynebacterium acetoglutamicum ATCC15806

[0070]Corynebacterium acetoacidophilum ATCC13870

[0071]Corynebacterium melassecola ATCC17965

[0072]Corynebacterium thermoaminogenes FERM BP-1539

[0073]Brevibacterium flavum ATCC14067

[0074]Brevibacterium lactofermentum ATCC13869 and

[0075]Brevibacterium divaricatum ATCC14020

[0076] or L-amino acid-producing mutants or strains prepared therefrom,as, for example the methionine-producing strain Corynebacteriumglutamicum ATCC21608.

[0077] Preferably, a bacterial strand with attenuated expression of metDgene products with MetD transcription regulator activity will improveamino acid yields at least 1%.

[0078] The inventors have isolated the new metD gene from C. glutamicum,which codes for MetD transcription regulator.

[0079] To isolate the metD gene or also other genes of C. glutamicum, agene library of this microorganism is first set up in Escherichia coli(E. coli). The setting up of gene libraries is described in generallyknown textbooks and handbooks. The textbook by Winnacker: Gene undKlone, Eine Einführung in die Gentechnologie [Genes and Clones, AnIntroduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany,1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50,495-508 (1987)). Bathe et al. (Molecular and General Genetics,252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032,which was set up with the aid of the cosmid vector SuperCos I (Wahl etal., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575). Börmann et al. (MolecularMicrobiology 6(3), 317-326)) (1992)) in turn describe a gene library ofC. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980,Gene 11, 291-298).

[0080] To prepare a gene library of C. glutamicum in E. coli, it is alsopossible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences,25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitablehosts are, in particular, those E. coli strains which are restriction-and recombination-defective, such as the strain DH5αmcr, which has beendescribed by Grant et al. (Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649).

[0081] The long DNA fragments cloned with the aid of cosmids or other λvectors can then in turn be subcloned and subsequently sequenced in theusual vectors which are suitable for DNA sequencing, such as isdescribed e.g. by Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

[0082] The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

[0083] The new DNA sequence of C. glutamicum which codes for the metDgene and which, as SEQ ID NO. 1, is a constituent of the presentinvention has been found in this manner. The amino acid sequence of thecorresponding protein has furthermore been derived from the present DNAsequence by the methods described above. The resulting amino acidsequence of the metD gene product is shown in SEQ ID NO. 2.

[0084] Coding DNA sequences, which result from SEQ ID NO. 1 by thedegeneracy of the genetic code, are also a constituent of the invention.In the same way, DNA sequences, which hybridize with SEQ ID NO. 1 orparts of SEQ ID NO. 1, are a constituent of the invention. Conservativeamino acid exchanges, such as e.g. exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. It isfurthermore known that changes on the N and/or C terminus of a proteincannot substantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences, which result in a corresponding mannerfrom SEQ ID NO. 2, are also a constituent of the invention.

[0085] In the same way, DNA sequences, which hybridize with SEQ ID NO. 1or parts of SEQ ID NO. 1, are a constituent of the invention. Finally,DNA sequences, which are prepared by the polymerase chain reaction (PCR)using primers, which result from SEQ ID NO. 1, are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

[0086] The skilled artisan will find instructions for identifying DNAsequences by means of hybridization can be found by the expert, interalia, in the handbook “The DIG System Users Guide for FilterHybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993)and in Liebl et al. (International Journal of Systematic Bacteriology41: 255-260 (1991)). The hybridization takes place under stringentconditions, that is to say only hybrids in which the probe and targetsequence, i.e. the polynucleotides treated with the probe, are at least70% identical are formed. It is known that the stringency of thehybridization, including the washing steps, is influenced or determinedby varying the buffer composition, the temperature and the saltconcentration. The hybridization reaction is preferably carried outunder a relatively low stringency compared with the washing steps(Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).

[0087] A 5×SSC buffer at a temperature of approx. 50° C.-68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides, which are less than 70% identicalto the sequence of the probe. Such hybrids are less stable and areremoved by washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2×SSC and optionallysubsequently 0.5×SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50° C.-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1×SSC. Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% identical to the sequence of the probe employed can beisolated by increasing the hybridization temperature stepwise from 50°C. to 68° C. in steps of approx. 1-2° C. Further instructions onhybridization are obtainable on the market in the form of so-called kits(e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany,Catalogue No. 1603558).

[0088] A skilled artisan will find instructions for amplification of DNAsequences with the aid of the polymerase chain reaction (PCR) can befound by the expert, inter alia, in the handbook by Gait:Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK,1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag,Heidelberg, Germany, 1994).

[0089] The inventors have shown that Coryneform bacteria produce aminoacids in an improved manner after attenuation of the metD gene.

[0090] To achieve attenuation, either the expression of the metD gene orthe catalytic properties of the enzyme protein can be reduced oreliminated. The two measures can optionally be combined.

[0091] The reduction in gene expression can take place by suitableculturing or by genetic modification (mutation) of the signal structuresof gene expression. Signal structures of gene expression are, forexample, repressor genes, activator genes, operators, promoters,attenuators, ribosome binding sites, the start codon and terminators.The expert can find information on this e.g. in WO 96/15246, in Boyd andMurphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil andChambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer(Biotechnology and Bioengineering 58: 191 (1998)), in Pátek et al.(Microbiology 142: 1297 (1996)), Vasicova et al. (Journal ofBacteriology 181: 6188 (1999)) and in known textbooks of genetics andmolecular biology, such as e.g. the textbook by Knippers (“MolekulareGenetik [Molecular Genetics]”, 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995) or that by Winnacker (“Gene und Klone [Genesand Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

[0092] Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“DieThreonindehydratase aus Corynebacterium glutamicum: Aufhebung derallosterischen Regulation und Struktur des Enzyms [Threonine dehydratasefrom Corynebacterium glutamicum: Canceling the allosteric regulation andstructure of the enzyme]”, Reports from the Jülich Research Center,Jül-2906, ISSN09442952, Jülich, Germany, 1994). Summarizing descriptionscan be found in known textbooks of genetics and molecular biology, suchas e.g. that by Hagemann (“Allgemeine Genetik [General Genetics]”,Gustav Fischer Verlag, Stuttgart, 1986).

[0093] Possible mutations are transitions, transversions, insertions anddeletions. These mutations may be referred to as “missense mutations” or“nonsense mutations”, depending on the effect of the amino acid exchangeon the enzyme activity. Insertions or deletions of at least one basepair (bp) in a gene lead to frame shift mutations, as a consequence ofwhich incorrect amino acids are incorporated or translation isinterrupted prematurely. Deletions of several codons typically lead to acomplete loss of the enzyme activity. Instructions on generation of suchmutations are prior art and can be found in known textbooks of geneticsand molecular biology, such as e.g. the textbook by Knippers(“Molekulare Genetik [Molecular Genetics]”, 6th edition, Georg ThiemeVerlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone[Genes and Clones]”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990)or that by Hagemann (“Allgemeine Genetik [General Genetics]”, GustavFischer Verlag, Stuttgart, 1986).

[0094] A common method of mutating genes of C. glutamicum is the methodof “gene disruption” and “gene replacement” described by Schwarzer andPühler (Bio/Technology 9, 84-87 (1991)).

[0095] In the method of gene disruption a central part of the codingregion of the gene of interest is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal ofBacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516). The plasmid vector, which contains the central part ofthe coding region of the gene, is then transferred into the desiredstrain of C. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)). Methods fortransformation are described, for example, by Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used, for example, by Fitzpatrick etal. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) toeliminate the recA gene of C. glutamicum.

[0096] In the method of “gene replacement”, a mutation, such as e.g. adeletion, insertion or base exchange, is established in vitro in thegene of interest. The allele prepared is in turn cloned in a vector,which is not replicative for C. glutamicum, and this is then transferredinto the desired host of C. glutamicum by transformation or conjugation.After homologous recombination by means of a first “cross-over” eventwhich effects integration and a suitable second “cross-over” event whicheffects excision in the target gene or in the target sequence, theincorporation of the mutation or of the allele is achieved. This methodwas used, for example, by Peters-Wendisch et al. (Microbiology 144,915-927 (1998)) to eliminate the pyc gene of C. glutamicum by adeletion.

[0097] A deletion, insertion or a base exchange can be incorporated intothe metD gene in this manner.

[0098] In addition, it may be advantageous for the production of L-aminoacids to enhance, in particular over-express, one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the attenuation ofthe metD gene.

[0099] The use of endogenous genes is in general preferred. The term“endogenous genes” or “endogenous nucleotide sequences” is understood tomean the genes or nucleotide sequences present in the population of aspecies.

[0100] Thus, for example, for the preparation of L-amino acids, inaddition to the attenuation of the metD gene at the same time one ormore of the genes chosen from the group consisting of:

[0101] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0102] the tpi gene which codes for triose phosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0103] the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0104] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-09224661, WO 01/70995),

[0105] the pyc gene which codes for pyruvate carboxylase (EP-A-1083225),

[0106] the lysC gene which codes for a feed-back resistant aspartatekinase (Accession No.P26512; EP-B-0387527; EP-A-0699759; WO 00/63388),

[0107] the hom gene which codes for homoserine dehydrogenase (EP-A0131171),

[0108] the metA gene which codes for homoserine O-acetyltransferase(ACCESSION Number AF052652),

[0109] the metB gene which codes for cystathionine gamma-synthase(ACCESSION Number AF126953),

[0110] the aecD gene which codes for cystathionine gamma-lyase(ACCESSION Number M89931),

[0111] the metY gene which codes for O-acetylhomoserine sulfhydrylase(DE 10043334, DSM 13556),

[0112] the glyA gene which codes for serine hydroxymethyltransferase(JP-A-08107788),

[0113] may be enhanced and, in particular, over-expressed.

[0114] Furthermore, it may be advantageous for the production of aminoacids, in addition to the attenuation of the metD gene, at the same timefor one or more of the genes chosen from the group consisting of:

[0115] the pck gene which codes for phosphoenol pyruvate carboxykinase(EP-A-1094111),

[0116] the pgi gene which codes for glucose 6-phosphate isomerase(EP-A-1087015, WO 01/07626),

[0117] the poxB gene which codes for pyruvate oxidase (EP-A-1096013),

[0118] the thrB gene which codes for homoserine kinase (ACCESSION NumberP08210),

[0119] the thrC gene which codes for threonine synthase (ACCESSIONNumber P23669),

[0120] the metk gene which codes for methionine adenosyltransferase(ACCESSION Number AJ290443)

[0121] the ddh gene which codes for meso-diaminopimelate D-dehydrogenase(ACCESSION Number Y00151),

[0122] to be attenuated and, in particular, for the expression thereofto be reduced.

[0123] In addition to the attenuation of the metD gene it mayfurthermore be advantageous for the production of amino acids toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0124] The invention also provides the microorganisms prepared accordingto the invention, and these can be cultured continuously ordiscontinuously in the batch process (batch culture) or in the fed batch(feed process) or repeated fed batch process (repetitive feed process)for the purpose of production of L-amino acids. A summary of knownculture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [BioprocessTechnology 1. Introduction to Bioprocess Technology (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktorenund periphere Einrichtungen [Bioreactors and Peripheral Equipment](Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0125] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

[0126] The substances:

[0127] (a) sugars and carbohydrates, such as e.g. glucose, sucrose,lactose, fructose, maltose, molasses, starch and cellulose,

[0128] (b) oils and fats, such as, soya oil, sunflower oil, groundnutoil and coconut fat,

[0129] (c) fatty acids, such as palmitic acid, stearic acid and linoleicacid,

[0130] (d) alcohols, such as glycerol and ethanol, and

[0131] (e) organic acids, such as acetic acid, may be used individually,or as a mixture, as the source of carbon.

[0132] The substances:

[0133] (a) Organic nitrogen-containing compounds, such as peptones,yeast extract, meat extract, malt extract, corn steep liquor, soya beanflour and urea, or

[0134] (b) inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,

[0135] can be used be used individually, or as a mixture, as the sourceof nitrogen.

[0136] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus.

[0137] The culture medium must furthermore comprise salts of metals,such as magnesium sulfate or iron sulfate, which are necessary forgrowth.

[0138] Essential growth substances, such as amino acids and vitamins,can be employed in addition to the above-mentioned substances. Suitableprecursors can moreover be added to the culture medium. The startingsubstances mentioned can be added to the culture in the form of a singlebatch, or can be fed in during the culture in a suitable manner.

[0139] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture.

[0140] Antifoams, such as, for example, fatty acid polyglycol esters,can be employed to control the development of foam. Suitable substanceshaving a selective action, such as, for example, antibiotics, can beadded to the medium to maintain the stability of plasmids. To maintainaerobic conditions, oxygen or oxygen-containing gas mixtures, such asair, are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of the desired product has formed. This targetis usually reached within 10 hours to 160 hours.

[0141] The fermentation broth prepared in this manner, in particularcontaining L-methionine, is then further processed. Depending onrequirements, all or some of the biomass can be removed from thefermentation broth by separation methods. Examples of such separationmethods are centrifugation, filtration, decanting or a combinationthereof. Alternatively, the biomass can be left completely in thefermentation broth. This broth is can optionally be thickened orconcentrated by known methods. Examples of such thickening orconcentrating methods include conventional methods such as evaporation,reverse osmosis, or by nanofiltration. Examples of instruments that canbe used in evaporation processes include methods a rotary evaporator,thin film evaporator, and falling film evaporator. This thickened orconcentrated fermentation broth can then be worked up. Examples ofmethods used to work up the thickened or concentrated fermentation brothinclude freeze drying, spray drying, spray granulation or by otherprocesses. Optionally, the fermentation broth can be worked up to yielda preferably free-flowing, finely divided powder.

[0142] The free-flowing, finely divided powder can be converted bysuitable compacting or granulating processes. Preferably, the powder canbe converted into a coarse-grained, readily free-flowing, storable andlargely dust-free product. During granulation or compaction, it isadvantageous to employ conventional organic or inorganic auxiliarysubstances or carriers. Examples of such organic or inorganic auxiliarysubstances or carriers include starch, gelatin, cellulose derivatives orsimilar substances. Further, these substances can be used as binders,gelling agents or thickeners in foodstuffs or feedstuffs processing.Further examples of these substances include silicas, silicates orstearates.

[0143] “Free-flowing” is understood as meaning powders which flowunimpeded out of the vessel with the opening of 5 mm (millimeters) of aseries of glass outflow vessels with outflow openings of various sizes(Klein, Seifen, Öle, Fette, Wachse 94, 12 (1968)).

[0144] As described here, “finely divided” means a powder with apredominant content, i.e. >50%, with a particle size of from 20 to 200μm diameter. The ranges for the particle size include all specificvalues and subranges therebetween, such as 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, and 190 μm diameter.“Coarse-grained” means products with a predominant content, i.e. >50%,with a particle size of 200 to 2000 μm diameter. The ranges for theparticle size include all specific values and subranges therebetween,such as 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, and 1900 μm diameter. In this context,“dust-free” means that the product contains only small contents, i.e.<5%, with particle sizes of less than 20 μm diameter. The particle sizedetermination can be carried out with methods of laser diffractionspectrometry. The corresponding methods are described in the textbook on“TeilchengröβBenmessung in der Laborpraxis” by R. H. Müller and R.Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or inthe textbook “Introduction to Particle Technology” by M. Rhodes, VerlagWiley & Sons (1998).

[0145] “Storable” in the context of this invention means a product whichcan be stored for up to 120 days, preferably up to 52 weeks,particularly preferably 60 months, without a substantial loss, i.e. <5%,of methionine.

[0146] Alternatively, however, the product can be absorbed onto anorganic or inorganic carrier substance which is known and conventionalin feedstuffs processing. Examples of such organic or inorganic carriersubstances include silicas, silicates, grits, brans, meals, starches,sugars or others. Further, the product simultaneously or subsequentlymixed and/or stabilized with conventional thickeners and/or binders.Examples of uses and processes in this context are described in theliterature (Die Müihle+Mischfuttertechnik 132 (1995) 49, page 817).

[0147] Finally, the product can be brought into a state in which it isstable to digestion by animal stomachs, in particular the stomach ofruminants, by coating processes, i.e. coating. Examples of suchconventional coating processes include those that use film-formingagents. Examples of film-forming agents include metal carbonates,silicas, silicates, alginates, stearates, starches, gums and celluloseethers, which are described in DE-C-4100920.

[0148] If the biomass is separated off during the process, furtherinorganic solids which can be optionally added during the fermentationcan be optionally removed. In addition, the animal feedstuffs additiveaccording to the invention can optionally comprise a predominantproportion, i.e. >50%, of further substances. Examples of such furthersubstances include organic substances which can be optionally formedand/or added and are optionally present in solution in the fermentationbroth, because they have optionally not been separated off by suitableprocesses.

[0149] In one aspect of the invention, the biomass can be separated offto the extent of up to 70%, preferably up to 80%, preferably up to 90%,preferably up to 95%, and particularly preferably up to 100%. In anotheraspect of the invention, up to 20% of the biomass, preferably up to 15%,preferably up to 10%, preferably up to 5%, particularly preferably nobiomass is separated off.

[0150] Examples of the above-mentioned organic substances includeorganic by-products. Organic by-products can be optionally produced, inaddition to the L-methionine, and can be optionally discharged by themicroorganisms employed in the fermentation. Examples of organicby-products include L-amino acids chosen from the group consisting ofL-valine, L-threonine, L-alanine or L-tryptophan. Further examples oforganic by-products include vitamins chosen from the group consisting ofvitamin B1 (thiamine), vitamin B2 (riboflavin),vitamin B5 (pantothenicacid), vitamin B6 (pyridoxine), vitamin B12 (cyanocobalamin), nicotinicacid/nicotinamide and vitamin E (tocopherol). Even further examples oforganic by-products include organic acids. Examples of organic acids arethose that contain one to three carboxyl groups. Examples of organicacids containing one to three carboxyl groups include acetic acid,lactic acid, citric acid, malic acid and/or fumaric acid. Finally,Examples of organic by-products include sugars. Examples of sugarsinclude such trehalose. These compounds are optionally desired if theyimprove the nutritional value of the product.

[0151] Organic substances, including L-methionine and/or D-methionineand/or the racemic mixture D,L-methionine, can optionally be addedduring a suitable process step. Organic substances can be in many forms.Examples of such forms include concentrate and/or pure substance insolid and/or liquid form. These organic substances mentioned canoptionally be added individually or as mixtures to the resulting orconcentrated fermentation broth, or also optionally during the drying orgranulation process. It is likewise possible to optionally add anorganic substance or a mixture of several organic substances to thefermentation broth and a optionally add further organic substance or afurther mixture of several organic substances during a later processstep. Examples of such as later step can include a granulation step.

[0152] The product described above is suitable as a feedstuffs additive,i.e. feed additive, for animal nutrition.

[0153] The L-methionine content of the animal feedstuffs additive isconventionally 1 wt. % to 80 wt. %, preferably 2 wt. % to 80 wt. %,particularly preferably 4 wt. % to 80 wt. %, and very particularlypreferably 8 wt. % to 80 wt. %, based on the dry weight of the animalfeedstuffs additive. Contents of 1 wt. % to 60 wt. %, 2 wt. % to 60 wt.%, 4 wt. % to 60 wt. %, 6 wt. % to 60 wt. %, 1 wt. % to 40 wt. %, 2 wt.% to 40 wt. % or 4 wt. % to 40 wt. % are likewise possible. The rangesfor content of the animal feedstuffs additive include all specificvalues and subranges therebetween, such 2, 4, 6, 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, and 75 wt. %. The water content of thefeedstuffs additive is conventionally up to 5 wt. %, preferably up to 4wt. %, and particularly preferably less than 2 wt. %.

[0154] An animal feedstuffs additive according to the present inventioncan comprise 1 wt. % to 80 wt. % L-methionine, D-methionine,D,L-methionine, or a mixture thereof with 1 to 40 wt. % L-lysine,D-lysine or D,L-lysine, based on the dry weight of the animal feedstuffsadditive. The ranges for content of L-methionine, D-methionine,D,L-methionine, or a mixture thereof with L-lysine, D-lysine orD,L-lysine in the animal feedstuffs additive include all specific valuesand subranges therebetween, such 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, and 75 wt. %. The ranges for content ofL-lysine, D-lysine or D,L-lysine in the mixture with L-methionine,D-methionine, D,L-methionine in the animal feedstuffs additive includeall specific values and subranges therebetween, such 2, 4, 6, 8, 10, 1015, 20, 25, 30, and 35 wt. %.

[0155] The invention accordingly also provides a process for thepreparation of an L-methionine-containing animal feedstuffs additivefrom fermentation broths, which comprises the steps:

[0156] h) culturing an L-methionine-producing microorganism in afermentation medium;

[0157] i) concentrating the L-methionine-containing fermentation broth;

[0158] j) removing an amount of from 0 to 100 wt. % of the biomassformed during the fermentation; and

[0159] k) drying of the fermentation broth obtained according to a)and/or b) to obtain the animal feedstuffs additive in the desired powderor granule form.

[0160] The concentrating step b) of the above-mentioned process includesthe removal of substances from the L-methionine-containing fermentationbroth. Examples of such substances that can be removed include water.

[0161] If desired, one or more of the following steps can furthermore becarried out in the process according to the invention:

[0162] l) adding at least one organic substances, including L-methionineand/or D-methionine and/or the racemic mixture D,L-methionine, to theproducts obtained according to a), b) and/or c);

[0163] m) adding auxiliary substances chosen from the group consistingof silicas, silicates, stearates, grits and bran to the substancesobtained according to a) to d) for stabilization and to increase thestorability; or

[0164] n) converting the substances obtained according to a) to e) intoa form stable to the animal stomach, in particular rumen, by coatingwith film-forming agents.

[0165] The analysis of L-methionine can be carried out by ion exchangechromatography with subsequent ninhydrin derivation, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190).

[0166] Methods for the determination of L-amino acids are known from theprior art. The analysis can thus be carried out, for example, asdescribed by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) byanion exchange chromatography with subsequent ninhydrin derivation, orit can be carried out by reversed phase HPLC, for example as describedby Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

[0167] The process according to the invention is used for fermentativepreparation of amino acids.

[0168] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatmentwere carried out by the method of Sambrook et al. (Molecular Cloning. ALaboratory Manual, 1989, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA). Methods for transformation of Escherichiacoli are also described in this handbook.

[0169] The composition of the usual nutrient media, such as LB or TYmedium, can also be found in the handbook by Sambrook et al.

[0170] The present invention is explained in more detail with the aid ofthe following embodiment examples.

EXAMPLES

[0171] The abbreviations and designations used have the followingmeaning: oriV ColE1-similar origin from pMB1 sacB sacB gene coding forthe protein levansucrase KmR Kanamycin resistance HindIII Cleavage siteof the restriction enzyme HindIII SwaI Cleavage site of the restrictionenzyme SwaI RP4mob RP4 mobilization site metD del Cloned deletionderivative for metD

Example 1

[0172] Preparation of a Genomic Cosmid Gene Library from C. glutamicumATCC 13032

[0173] Chromosomal DNA from C. glutamicum ATCC 13032 is isolated asdescribed by Tauch et al. (1995, Plasmid 33:168-179) and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNAfragments are dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP, Codeno. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al.(1987), Proceedings of the National Academy of Sciences, USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) is cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

[0174] The cosmid DNA is then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner is mixed with thetreated ATCC13032 DNA and the batch is treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture is then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

[0175] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Res. 16:1563-1575) the cells are taken up in 10 mM MgSO₄and mixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library are carried out as described by Sambrooket al. (1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190)+100 mg/l ampicillin. After incubation overnight at 37° C.,recombinant individual clones are selected.

Example 2

[0176] Isolation and Sequencing of the metD Gene

[0177] The cosmid DNA of an individual colony is isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments are dephosphorylated with shrimp alkaline phosphatase (RocheMolecular Biochemicals, Mannheim, Germany, Product Description SAP,Product No. 1758250). After separation by gel electrophoresis, thecosmid fragments in the size range of 1500 to 2000 bp are isolated withthe QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,Germany).

[0178] The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, Holland, Product Description Zero BackgroundCloning Kit, Product No. K2500-01) is cleaved with the restrictionenzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionBamHI, Product No. 27-0868-04). The ligation of the cosmid fragments inthe sequencing vector pZero-1 is carried out as described by Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture is thenelectroporated (Tauch et al. 1994, FEMS Microbiol. Letters, 123:343-7)into the E. coli strain DH5αmcr (Grant, 1990, Proceedings of theNational Academy of Sciences, U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

[0179] The plasmid preparation of the recombinant clones is carried outwith a Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing is carried out by the dideoxy chain-stopping method of Sangeret al. (1977, Proceedings of the National Academies of Sciences, U.S.A.,74:5463-5467) with modifications according to Zimmermann et al. (1990,Nucleic Acids Research, 18:1067). The “RR dRhodamin Terminator CycleSequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction are carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

[0180] The raw sequence data obtained are then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0. The individual sequences of the pZero1 derivatives areassembled to a continuous contig. The computer-assisted coding regionanalysis is prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch 14:217-231). Further analyses are carried out with the “BLASTsearch programs” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402) against the non-redundant databank of the “National Centerfor Biotechnology Information” (NCBI, Bethesda, Md., USA).

[0181] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of the nucleotide sequence shows an open reading frame of 711bp, which is called the metD gene. The metD gene codes for a polypeptideof 236 amino acids.

[0182] The DNA sections lying upstream and downstream of SEQ ID No. 1were identified in the same way, these sections being shown in SEQ IDNo. 3 and SEQ ID No. 4. The metD gene region extended by SEQ ID No. 3and SEQ ID No. 4 is shown in SEQ ID No. 5.

Example 3

[0183] Incorporation of a Deletion into the metD Gene

[0184] For this, chromosomal DNA is isolated from the strain ATCC13032by the method of Tauch et al. (Plasmid 33:168-179 (1995)). On the basisof the sequence of the metD gene known for C. glutamicum from example 2,the oligonucleotides described below are chosen for generation of themetD deletion allele by means of the polymerase chain reaction (PCR) bythe gene Soeing method (Horton, Molecular Biotechnology 3: 93-98(1995)). Primer metD-DelA (see also SEQ ID No.6): 5′-GAT CTA AAG CTT-GCCTCT CCA ATC TCC ACT GA-3′ Primer metD-DelB (see also SEQ ID No.7):5′-ATT GAG TAG TCC GCA GGT GG-ATT TAA AT-AAT CCA CAG GCA AGT CTA GC-3′Primer metD-DelC (see also SEQ ID No.8): 5′-GCT AGA CTT GCC TGT GGATT-ATT TAA AT-CCA CCT GCG GAC TAC TCA AT-3′ Primer metD-DelD (see alsoSEQ ID No.9): 5′-GAT CTA AAG CTT-GAT GTC CAT GTA CCG CAG C-3′

[0185] The primers shown are synthesized by MWG Biotech (Ebersberg,Germany) and the PCR reaction is carried out using Pfu polymerase(Stratagene, Product. No. 600135, La Jolla, USA) and a PTC 100Thermocycler (M J Research Inc., Waltham, USA).

[0186] The primers metD-DelA and metD-DelD contain in each case aninserted cleavage site for the restriction enzyme HindIII, and theprimers metD-DelB and metD-DelC an inserted cleavage site for therestriction enzyme SwaI, which are marked by underlining in thenucleotide sequence shown above.

[0187] The primer metD-DelB is composed of two regions of the nucleotidesequence, one of which bonds in the coding sequence of metD to thenucleotides 707 to 688, and the other bonds to the “upstream” region infront of the start codon of metD. Both regions are divided by theinserted Swal restriction enzyme site. The Primer metD-DelC is reversecomplementary to the Primer metD-DelB.

[0188] With the aid of the polymerase chain reaction the primersmetD-DelA and metD-DelB enable the amplification of a 573-bp DNAfragment and the Primers metD-DelC and metD-DelD enable theamplification of a 651 bp DNA fragment. The amplificates are examined bysubsequent agarose-gel electrophoresis in an 0.8% agarose-gel, isolatedfrom the agarose-gel with the High Pure PCR Product Purification Kit(Product No. 1732676, Roche Diagnostics GmbH, Mannheim, Deutschland),and used together as a DNA template in another PCR reaction using theprimers metD-DelA and metD-DelD. This results in the production of themetD deletion derivative, 1177 bp in size (see also SEQ ID No. 10).

[0189] The amplified product is subsequently examined in a 0.8%agarose-gel.

Example 4

[0190] 4.1 Construction of the Exchange Vector pK18mobsacBmetD del

[0191] The metD deletion derivative obtained in example 3 is cleavedwith the restriction endonuclease HindIII, after examination in a 0.8%agarose-gel isolated from the agarose gel with the High Pure PCR Productpurification Kit (Product No. 1732676, Roche Diagnostics GmbH, Mannheim,Deutschland) and used for ligation with the mobilizable cloning vectorpK18mobsacB described by Schäfer et al., Gene, 14, 69-73 (1994). Thevector pK18mobsacB was cleaved beforehand with the restriction enzymeHindIll and subsequently dephosphorylated with shrimp alkalinephosphatase (Roche Diagnostics GmbH, Mannheim, Germany, ProductDescription SAP, Product No. 1758250). The prepared vector is then mixedwith the metD deletion derivative and treated with T4 DNA ligase(Amersham-Pharmacia, Freiburg, Germany).

[0192] The E. coli strain DH5αmcr (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87: 4645-4649) is thenelectroporated with the ligation batch (Hanahan, In. DNA cloning. Apractical approach. Vol. 1. ILR-Press, Cold Spring Harbor, N.Y., 1989).Selection of plasmid-carrying cells is made by plating out thetransformation batch on LB agar (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor, N.Y., 1989), whichhas been supplemented with 25 mg/l kanamycin.

[0193] Plasmid DNA is isolated from a transformant with the aid of theQIAprep Spin Miniprep Kit from Qiagen and verified by cutting with therestriction enzymes HindIII and SwaI. The plasmid is calledpK18mobsacBmetD del and is shown in FIG. 1. The strain is called E. coliDH5αmcr/pK18mobsacBmetD del.

[0194] 4.2 Deletion Mutagenesis of the metD Gene in the C. glutamicumStrain ATCC13032

[0195] The vector pK18mobsacBmetD del mentioned in example 4.1 iselectroporated by the electroporation method of Tauch et al.,(1989 FEMSMicrobiology Letters 123: 343-347) in the strain C. glutamicumATCC13032. The vector cannot replicate independently in ATCC13032 and isretained in the cell only if it has integrated into the chromosome.Selection of clones with integrated pK18mobsacmetD del is carried out byplating out the electroporation batch on LB agar (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor,N.Y., 1989), which has been supplemented with mg/l kanamycin. Cloneswhich had grown on are plated out on LB agar plates with 25 mg/lkanamycin and incubated for 16 hours at 33° C.

[0196] To achieve excision of the plasmid together with the completechromosomal copy of the metD gene, the clones are incubatedunselectively overnight in LB medium and then cultured on LB agar with10% sucrose. The plasmid pK18mobsacB contains a copy of the sacB gene,which converts sucrose into levan sucrose, which is toxic to C.glutamicum. Only those clones in which the integrated pK18mobsacBmetDdel has been excised again therefore grow on LB agar with sucrose. Inthe excision, together with the plasmid either the complete chromosomalcopy of the metD gene can be excised, or the metD deletion derivative.

[0197] To demonstrate that the metD gene is deleted in the chromosome,the plasmid pK18mobsacBmetD del is marked by the method of “The DIGSystem Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH(Mannheim, Germany, 1993) using the DIG hybridization kit fromBoehringer. Chromosomal DNA of a potential deletion mutant is isolatedby the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994))and in each case cleaved with the restriction enzymes HindIII and SwaIin separate batches. The fragments formed are separated by agarose gelelectrophoresis and hybridized at 68° C. with the Dig hybridization kitfrom Boehringer. With the aid of the fragments formed, it can be shownthat the strain ATCC13032 has lost its copy of the metD gene, andinstead carries the deleted allele.

[0198] The strain is called C. glutamicum ATCC13032ΔmetD.

Example 5

[0199] Production of Methionine

[0200] The C. glutamicum strain ATCC13032ΔmetD obtained in example 4 iscultured in a nutrient medium suitable for the production of methionineand the methionine content in the culture supernatant is determined.

[0201] For this, the strain is first incubated on a brain-heart agarplate for 24 hours at 33° C. Starting from this agar plate culture, apreculture is seeded (10 ml medium in a 100 ml conical flask). Themedium MM is used as the medium for the preculture. Medium MM CSL (cornsteep liquor) 5 g/l MOPS (morpholinopropanesulfonic acid) 20 g/l Glucose(autoclaved separately) 50 g/l Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/lMgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/lMnSO₄ * H₂O 5.0 mg/l Biotin (sterile-filtered) 0.01 mg/l Vitamin B12(sterile-filtered) 0.02 mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/lCaCO₃ 25 g/l

[0202] The CSL, MOPS and the salt solution are brought to pH 7 withaqueous ammonia and autoclaved. The sterile substrate and vitaminsolutions are then added, as well as the CaCO₃ autoclaved in the drystate.

[0203] The preculture is incubated for 16 hours at 33° C. at 240 rpm ona shaking machine. A main culture is seeded from this preculture suchthat the initial OD (660 nm) of the main culture was 0.1. Medium MM isalso used for the main culture.

[0204] Culturing is carried out in a 10 ml volume in a 100 ml conicalflask with baffles. Culturing is carried out at 33° C. and 80%atmospheric humidity.

[0205] After 72 hours, the OD is determined at a measurement wavelengthof 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). Theamount of methionine formed is determined with an amino acid analyzerfrom Eppendorf-BioTronik (Hamburg, Germany) by ion exchangechromatography and post-column derivation with ninhydrin detection.

[0206] The result of the experiment is shown in Table 1. TABLE 1 ODMethionine Strain (660 nm) mg/l ATCC13032 12.2  3 ATCC13032ΔmetD 14.8 20

[0207] Numerous modifications and variations on the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

[0208] The present application claims priority to German Application No.DE 10126164.0, filed May 30, 2001. The entire content of thisapplication is incorporated herein by reference.

1 11 1 1322 DNA Corynebacterium glutamicum CDS (314)..(1021) 1agattaaagg cactgatgct cagcaaggaa ttttgctgaa catagtcgtc ggtattatcg 60gtggtttgtt aggcggctgg ctgcttggaa tcttcggagt ggatgttgcc ggtggcggct 120tgatcttcag cttcatcaca tgtctgattg gtgctgtcat tttgctgacg atcgtgcagt 180tcttcactcg gaagaagtaa tctgctttaa atccgtaggg cctgttgata tttcgatatc 240aacaggcctt ttggtcattt tggggtggaa aaagcgctag acttgcctgt ggattaaaac 300tatacgaacc ggt ttg tct ata ttg gtg tta gac agt tcg tcg tat ctt 349 LeuSer Ile Leu Val Leu Asp Ser Ser Ser Tyr Leu 1 5 10 gaa aca gac caa cccgaa agg acg tgg ccg aac gtg gct gct agc gct 397 Glu Thr Asp Gln Pro GluArg Thr Trp Pro Asn Val Ala Ala Ser Ala 15 20 25 tca ggc aag agt aaa acaagt gcc ggg gca aac cgt cgt cgc aat cga 445 Ser Gly Lys Ser Lys Thr SerAla Gly Ala Asn Arg Arg Arg Asn Arg 30 35 40 cca agc ccc cga cag cgt ctcctc gat agc gca acc aac ctt ttc acc 493 Pro Ser Pro Arg Gln Arg Leu LeuAsp Ser Ala Thr Asn Leu Phe Thr 45 50 55 60 aca gaa ggt att cgc gtc atcggt att gat cgt atc ctc cgt gaa gct 541 Thr Glu Gly Ile Arg Val Ile GlyIle Asp Arg Ile Leu Arg Glu Ala 65 70 75 gac gtg gcg aag gcg agc ctc tattcc ctt ttc gga tcg aag gac gcc 589 Asp Val Ala Lys Ala Ser Leu Tyr SerLeu Phe Gly Ser Lys Asp Ala 80 85 90 ttg gtt att gca tac ctg gag aac ctcgat cag ctg tgg cgt gaa gcg 637 Leu Val Ile Ala Tyr Leu Glu Asn Leu AspGln Leu Trp Arg Glu Ala 95 100 105 tgg cgt gag cgc acc gtc ggt atg aaggat ccg gaa gat aaa atc atc 685 Trp Arg Glu Arg Thr Val Gly Met Lys AspPro Glu Asp Lys Ile Ile 110 115 120 gcg ttc ttt gat cag tgc att gag gaagaa cca gaa aaa gat ttc cgc 733 Ala Phe Phe Asp Gln Cys Ile Glu Glu GluPro Glu Lys Asp Phe Arg 125 130 135 140 ggc tcg cac ttt cag aat gcg gctagt gag tac cct cgc ccc gaa act 781 Gly Ser His Phe Gln Asn Ala Ala SerGlu Tyr Pro Arg Pro Glu Thr 145 150 155 gat agc gaa aag ggc att gtt gcagca gtg tta gag cac cgc gag tgg 829 Asp Ser Glu Lys Gly Ile Val Ala AlaVal Leu Glu His Arg Glu Trp 160 165 170 tgt cat aag act ctg act gat ttgctc act gag aag aac ggc tac cca 877 Cys His Lys Thr Leu Thr Asp Leu LeuThr Glu Lys Asn Gly Tyr Pro 175 180 185 ggc acc acc cag gcg aat cag ctgttg gtg ttc ctt gat ggt gga ctt 925 Gly Thr Thr Gln Ala Asn Gln Leu LeuVal Phe Leu Asp Gly Gly Leu 190 195 200 gct gga tct cga ttg gtc cac aacatc agt cct ctt gag acg gct cgc 973 Ala Gly Ser Arg Leu Val His Asn IleSer Pro Leu Glu Thr Ala Arg 205 210 215 220 gat ttg gct cgg cag ttg ttgtcg gct cca cct gcg gac tac tca att 1021 Asp Leu Ala Arg Gln Leu Leu SerAla Pro Pro Ala Asp Tyr Ser Ile 225 230 235 tagtttcttc attttccgaaggggtatctt cgttggggga ggcgtcgata agccccttct 1081 ttttagcttt aacctcagcgcgacgctgct ttaagcgctg catggcggcg cggttcattt 1141 cacgttgcgt ttcgcgcctcttgttcgcga tttctttgcg ggcctgtttt gcttcgttga 1201 tttcggcagt acgggttttggtgagttcca cgtttgttgc gtgaagcgtt gaggcgttcc 1261 atggggtgag aatcatcagggcgcggtttt tgcgtcgtgt ccacaggaag atgcgctttt 1321 c 1322 2 236 PRTCorynebacterium glutamicum 2 Leu Ser Ile Leu Val Leu Asp Ser Ser Ser TyrLeu Glu Thr Asp Gln 1 5 10 15 Pro Glu Arg Thr Trp Pro Asn Val Ala AlaSer Ala Ser Gly Lys Ser 20 25 30 Lys Thr Ser Ala Gly Ala Asn Arg Arg ArgAsn Arg Pro Ser Pro Arg 35 40 45 Gln Arg Leu Leu Asp Ser Ala Thr Asn LeuPhe Thr Thr Glu Gly Ile 50 55 60 Arg Val Ile Gly Ile Asp Arg Ile Leu ArgGlu Ala Asp Val Ala Lys 65 70 75 80 Ala Ser Leu Tyr Ser Leu Phe Gly SerLys Asp Ala Leu Val Ile Ala 85 90 95 Tyr Leu Glu Asn Leu Asp Gln Leu TrpArg Glu Ala Trp Arg Glu Arg 100 105 110 Thr Val Gly Met Lys Asp Pro GluAsp Lys Ile Ile Ala Phe Phe Asp 115 120 125 Gln Cys Ile Glu Glu Glu ProGlu Lys Asp Phe Arg Gly Ser His Phe 130 135 140 Gln Asn Ala Ala Ser GluTyr Pro Arg Pro Glu Thr Asp Ser Glu Lys 145 150 155 160 Gly Ile Val AlaAla Val Leu Glu His Arg Glu Trp Cys His Lys Thr 165 170 175 Leu Thr AspLeu Leu Thr Glu Lys Asn Gly Tyr Pro Gly Thr Thr Gln 180 185 190 Ala AsnGln Leu Leu Val Phe Leu Asp Gly Gly Leu Ala Gly Ser Arg 195 200 205 LeuVal His Asn Ile Ser Pro Leu Glu Thr Ala Arg Asp Leu Ala Arg 210 215 220Gln Leu Leu Ser Ala Pro Pro Ala Asp Tyr Ser Ile 225 230 235 3 239 DNAArtificial Sequence Synthetic DNA 3 gcctctccaa tctccactga ggtacttaatccttccgggg aattcgggcg cttaaatcga 60 gaaattaggc catcaccttt taataacaatacaatgaata attggaatag gtcgacacct 120 ttggagcgga gccggttaaa attggcagcattcaccgaaa gaaaaggaga accacatgct 180 tgccctaggt tggattacat ggatcattattggtggtcta gctggttgga ttgcctcca 239 4 289 DNA Artificial SequenceSynthetic DNA 4 tttttgtttt gcgcggtaga tgtcgcgctg ctctaggtgg tgcactttgaaatcgtcggt 60 aagtgggtat ttgcgttcca aaatgaccat catgatgatt gtttggaggagcgtccacag 120 gttgttgctg acccaataga gtgcgattgc tgtggggaat ggtcctgtgaggccaaggga 180 cagtgggaag atcggcgcga ggatcgacat cacgatcatg aacttcagcatgccgttaga 240 gaatccggat gcgtaatcgt tggtttggaa gctgcggtac atggacatc 2895 1850 DNA Corynebacterium glutamicum CDS (553)..(1260) 5 gcctctccaatctccactga ggtacttaat ccttccgggg aattcgggcg cttaaatcga 60 gaaattaggccatcaccttt taataacaat acaatgaata attggaatag gtcgacacct 120 ttggagcggagccggttaaa attggcagca ttcaccgaaa gaaaaggaga accacatgct 180 tgccctaggttggattacat ggatcattat tggtggtcta gctggttgga ttgcctccaa 240 gattaaaggcactgatgctc agcaaggaat tttgctgaac atagtcgtcg gtattatcgg 300 tggtttgttaggcggctggc tgcttggaat cttcggagtg gatgttgccg gtggcggctt 360 gatcttcagcttcatcacat gtctgattgg tgctgtcatt ttgctgacga tcgtgcagtt 420 cttcactcggaagaagtaat ctgctttaaa tccgtagggc ctgttgatat ttcgatatca 480 acaggccttttggtcatttt ggggtggaaa aagcgctaga cttgcctgtg gattaaaact 540 atacgaaccg gtttg tct ata ttg gtg tta gac agt tcg tcg tat ctt gaa 591 Leu Ser Ile LeuVal Leu Asp Ser Ser Ser Tyr Leu Glu 1 5 10 aca gac caa ccc gaa agg acgtgg ccg aac gtg gct gct agc gct tca 639 Thr Asp Gln Pro Glu Arg Thr TrpPro Asn Val Ala Ala Ser Ala Ser 15 20 25 ggc aag agt aaa aca agt gcc ggggca aac cgt cgt cgc aat cga cca 687 Gly Lys Ser Lys Thr Ser Ala Gly AlaAsn Arg Arg Arg Asn Arg Pro 30 35 40 45 agc ccc cga cag cgt ctc ctc gatagc gca acc aac ctt ttc acc aca 735 Ser Pro Arg Gln Arg Leu Leu Asp SerAla Thr Asn Leu Phe Thr Thr 50 55 60 gaa ggt att cgc gtc atc ggt att gatcgt atc ctc cgt gaa gct gac 783 Glu Gly Ile Arg Val Ile Gly Ile Asp ArgIle Leu Arg Glu Ala Asp 65 70 75 gtg gcg aag gcg agc ctc tat tcc ctt ttcgga tcg aag gac gcc ttg 831 Val Ala Lys Ala Ser Leu Tyr Ser Leu Phe GlySer Lys Asp Ala Leu 80 85 90 gtt att gca tac ctg gag aac ctc gat cag ctgtgg cgt gaa gcg tgg 879 Val Ile Ala Tyr Leu Glu Asn Leu Asp Gln Leu TrpArg Glu Ala Trp 95 100 105 cgt gag cgc acc gtc ggt atg aag gat ccg gaagat aaa atc atc gcg 927 Arg Glu Arg Thr Val Gly Met Lys Asp Pro Glu AspLys Ile Ile Ala 110 115 120 125 ttc ttt gat cag tgc att gag gaa gaa ccagaa aaa gat ttc cgc ggc 975 Phe Phe Asp Gln Cys Ile Glu Glu Glu Pro GluLys Asp Phe Arg Gly 130 135 140 tcg cac ttt cag aat gcg gct agt gag taccct cgc ccc gaa act gat 1023 Ser His Phe Gln Asn Ala Ala Ser Glu Tyr ProArg Pro Glu Thr Asp 145 150 155 agc gaa aag ggc att gtt gca gca gtg ttagag cac cgc gag tgg tgt 1071 Ser Glu Lys Gly Ile Val Ala Ala Val Leu GluHis Arg Glu Trp Cys 160 165 170 cat aag act ctg act gat ttg ctc act gagaag aac ggc tac cca ggc 1119 His Lys Thr Leu Thr Asp Leu Leu Thr Glu LysAsn Gly Tyr Pro Gly 175 180 185 acc acc cag gcg aat cag ctg ttg gtg ttcctt gat ggt gga ctt gct 1167 Thr Thr Gln Ala Asn Gln Leu Leu Val Phe LeuAsp Gly Gly Leu Ala 190 195 200 205 gga tct cga ttg gtc cac aac atc agtcct ctt gag acg gct cgc gat 1215 Gly Ser Arg Leu Val His Asn Ile Ser ProLeu Glu Thr Ala Arg Asp 210 215 220 ttg gct cgg cag ttg ttg tcg gct ccacct gcg gac tac tca att 1260 Leu Ala Arg Gln Leu Leu Ser Ala Pro Pro AlaAsp Tyr Ser Ile 225 230 235 tagtttcttc attttccgaa ggggtatctt cgttgggggaggcgtcgata agccccttct 1320 ttttagcttt aacctcagcg cgacgctgct ttaagcgctgcatggcggcg cggttcattt 1380 cacgttgcgt ttcgcgcctc ttgttcgcga tttctttgcgggcctgtttt gcttcgttga 1440 tttcggcagt acgggttttg gtgagttcca cgtttgttgcgtgaagcgtt gaggcgttcc 1500 atggggtgag aatcatcagg gcgcggtttt tgcgtcgtgtccacaggaag atgcgctttt 1560 ctttttgttt tgcgcggtag atgtcgcgct gctctaggtggtgcactttg aaatcgtcgg 1620 taagtgggta tttgcgttcc aaaatgacca tcatgatgattgtttggagg agcgtccaca 1680 ggttgttgct gacccaatag agtgcgattg ctgtggggaatggtcctgtg aggccaaggg 1740 acagtgggaa gatcggcgcg aggatcgaca tcacgatcatgaacttcagc atgccgttag 1800 agaatccgga tgcgtaatcg ttggtttgga agctgcggtacatggacatc 1850 6 236 PRT Corynebacterium glutamicum 6 Leu Ser Ile LeuVal Leu Asp Ser Ser Ser Tyr Leu Glu Thr Asp Gln 1 5 10 15 Pro Glu ArgThr Trp Pro Asn Val Ala Ala Ser Ala Ser Gly Lys Ser 20 25 30 Lys Thr SerAla Gly Ala Asn Arg Arg Arg Asn Arg Pro Ser Pro Arg 35 40 45 Gln Arg LeuLeu Asp Ser Ala Thr Asn Leu Phe Thr Thr Glu Gly Ile 50 55 60 Arg Val IleGly Ile Asp Arg Ile Leu Arg Glu Ala Asp Val Ala Lys 65 70 75 80 Ala SerLeu Tyr Ser Leu Phe Gly Ser Lys Asp Ala Leu Val Ile Ala 85 90 95 Tyr LeuGlu Asn Leu Asp Gln Leu Trp Arg Glu Ala Trp Arg Glu Arg 100 105 110 ThrVal Gly Met Lys Asp Pro Glu Asp Lys Ile Ile Ala Phe Phe Asp 115 120 125Gln Cys Ile Glu Glu Glu Pro Glu Lys Asp Phe Arg Gly Ser His Phe 130 135140 Gln Asn Ala Ala Ser Glu Tyr Pro Arg Pro Glu Thr Asp Ser Glu Lys 145150 155 160 Gly Ile Val Ala Ala Val Leu Glu His Arg Glu Trp Cys His LysThr 165 170 175 Leu Thr Asp Leu Leu Thr Glu Lys Asn Gly Tyr Pro Gly ThrThr Gln 180 185 190 Ala Asn Gln Leu Leu Val Phe Leu Asp Gly Gly Leu AlaGly Ser Arg 195 200 205 Leu Val His Asn Ile Ser Pro Leu Glu Thr Ala ArgAsp Leu Ala Arg 210 215 220 Gln Leu Leu Ser Ala Pro Pro Ala Asp Tyr SerIle 225 230 235 7 32 DNA Artificial Sequence Synthetic DNA 7 gatctaaagcttgcctctcc aatctccact ga 32 8 48 DNA Artificial Sequence Synthetic DNA 8attgagtagt ccgcaggtgg atttaaataa tccacaggca agtctagc 48 9 48 DNAArtificial Sequence Synthetic DNA 9 gctagacttg cctgtggatt atttaaatccacctgcggac tactcaat 48 10 31 DNA Artificial Sequence Synthetic DNA 10gatctaaagc ttgatgtcca tgtaccgcag c 31 11 1177 DNA Artificial SequenceSynthetic DNA 11 gatctaaagc ttgcctctcc aatctccact gaggtactta atccttccggggaattcggg 60 cgcttaaatc gagaaattag gccatcacct tttaataaca atacaatgaataattggaat 120 aggtcgacac ctttggagcg gagccggtta aaattggcag cattcaccgaaagaaaagga 180 gaaccacatg cttgccctag gttggattac atggatcatt attggtggtctagctggttg 240 gattgcctcc aagattaaag gcactgatgc tcagcaagga attttgctgaacatagtcgt 300 cggtattatc ggtggtttgt taggcggctg gctgcttgga atcttcggagtggatgttgc 360 cggtggcggc ttgatcttca gcttcatcac atgtctgatt ggtgctgtcattttgctgac 420 gatcgtgcag ttcttcactc ggaagaagta atctgcttta aatccgtagggcctgttgat 480 atttcgatat caacaggcct tttggtcatt ttggggtgga aaaagcgctagacttgcctg 540 tggattattt aaatccacct gcggactact caatttagtt tcttcattttccgaaggggt 600 atcttcgttg ggggaggcgt cgataagccc cttcttttta gctttaacctcagcgcgacg 660 ctgctttaag cgctgcatgg cggcgcggtt catttcacgt tgcgtttcgcgcctcttgtt 720 cgcgatttct ttgcgggcct gttttgcttc gttgatttcg gcagtacgggttttggtgag 780 ttccacgttt gttgcgtgaa gcgttgaggc gttccatggg gtgagaatcatcagggcgcg 840 gtttttgcgt cgtgtccaca ggaagatgcg cttttctttt tgttttgcgcggtagatgtc 900 gcgctgctct aggtggtgca ctttgaaatc gtcggtaagt gggtatttgcgttccaaaat 960 gaccatcatg atgattgttt ggaggagcgt ccacaggttg ttgctgacccaatagagtgc 1020 gattgctgtg gggaatggtc ctgtgaggcc aagggacagt gggaagatcggcgcgaggat 1080 cgacatcacg atcatgaact tcagcatgcc gttagagaat ccggatgcgtaatcgttggt 1140 ttggaagctg cggtacatgg acatcaagct ttagatc 1177

What is claimed is:
 1. An isolated polynucleotide sequence, whichencodes a polypeptide having the amino acid sequence of SEQ ID NO.
 2. 2.The isolated polynucleotide sequence of claim 1, wherein saidpolypeptide sequence has MetD transcription regulator activity.
 3. Avector comprising the isolated polynucleotide sequence of claim
 1. 4. Ahost cell comprising the isolated polynucleotide sequence of claim
 1. 5.The host cell of claim 4, which is a Coryneform bacterium.
 6. The hostcell of claim 4, which is a Coryneform bacterium selected from the groupconsisting of Corynebacterium glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriummelassecola, Corynebacterium thermoaminogenes, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 7. Amethod for detecting polynucleotides with at least 70% homology to thepolynucleotide of claim 1, comprising contacting a polynucleotide samplewith a polynucleotide comprising at least 15 consecutive nucleotides ofthe polynucleotide sequence of claim 1, or at least 15 consecutivenucleotides of the complement thereof.
 8. A method for producingpolynucleotides with at least 70% homology to the polynucleotide ofclaim 1, comprising contacting a polynucleotide sample with apolynucleotide comprising at least 15 consecutive nucleotides of thepolynucleotide sequence of claim 1, or at least 15 consecutivenucleotides of the complement thereof.
 9. A process for screening forpolynucleotide sequences, which encode a polypeptide having MetDtranscription regulator activity comprising (a) hybridizing the isolatedpolynucleotide to claim 1 to a polynucleotide sample to be screened; (b)expressing the polynucleotide to produce a polypeptide; (c) detectingthe presence or absence of MetD transcription regulator activity of thepolypeptide.
 10. The process according to claim 9, wherein saidhybridizing is performed with arrays, micro arrays, DNA chips, orcombinations thereof.
 11. A method for making MetD transcriptionregulator polypeptide, comprising (a) culturing the host cell of claim 4for a duration of time under conditions suitable for expression of MetDtranscription regulator polypeptide; and (b) collecting the MetDtranscription regulator polypeptide.
 12. An isolated polynucleotide,which comprises SEQ ID NO.
 1. 13. An isolated polynucleotide, which iscomplementary to the polynucleotide of claim
 12. 14. An isolatedpolynucleotide, which is at least 70% identical to the polynucleotide ofclaim
 12. 15. An isolated polynucleotide, which is at least 80%identical to the polynucleotide of claim
 12. 16. An isolatedpolynucleotide, which is at least 90% identical to the polynucleotide ofclaim
 12. 17. An isolated polynucleotide, which comprises at least 15consecutive nucleotides of the polynucleotide of claim
 12. 18. Anisolated polynucleotide, which hybridizes to the polynucleotide of claim12.
 19. The isolated polynucleotide of claim 12, which encodes apolypeptide having MetD transcription regulator activity.
 20. A vectorcomprising the isolated polynucleotide of claim
 12. 21. A host cellcomprising the isolated polynucleotide of claim
 12. 22. The host cell ofclaim 21, which is a Coryneform bacterium.
 23. The host cell of claim21, which is a Coryneform bacterium selected from the group consistingof Corynebacterium glutamicum, Corynebacterium acetoglutamicum,Corynebacterium acetoacidophilum, Corynebacterium melassecola,Corynebacterium thermoaminogenes, Brevibacterium flavum, Brevibacteriumlactofermentum, and Brevibacterium divaricatum.
 24. A process forscreening for polynucleotide sequences, which encode a polypeptidehaving MetD transcription regulator activity comprising (a) hybridizingthe isolated polynucleotide to claim 11 to a polynucleotide sample to bescreened; (b) expressing the polynucleotide to produce a polypeptide;(c) detecting the presence or absence of MetD transcription regulatoractivity of the polypeptide.
 25. A method for detecting polynucleotideswith at least 70% homology to the polynucleotide of claim 12, comprisingcontacting a polynucleotide sample with a polynucleotide comprising atleast 15 consecutive nucleotides of the polynucleotide sequence of claim12, or at least 15 consecutive nucleotides of the complement thereof.26. A method for producing polynucleotides with at least 70% homology tothe polynucleotide of claim 12, comprising contacting a polynucleotidesample with a polynucleotide comprising at least 15 consecutivenucleotides of the polynucleotide sequence of claim 12, or at least 15consecutive nucleotides of the complement thereof.
 27. A method formaking MetD transcription regulator polypeptide, comprising (a)culturing the host cell of claim 21 for a duration of time underconditions suitable for expression of MetD transcription regulatorpolypeptide; and (b) collecting the MetD transcription regulatorpolypeptide.
 28. A Coryneform bacterium, which comprises attenuatedexpression of the metD gene.
 29. The Coryneform bacterium of claim 28,wherein the metD gene comprises the polynucleotide sequence of SEQ IDNO.
 1. 30. Corynebacterium glutamicum strain ATCC13032deltametD. 31.Escherichia coli DH5αmcr/pK18mobsacBmetD del.
 32. A process forproducing L-amino acids comprising culturing a bacterial cell in amedium suitable for producing L-amino acids, wherein the bacterial cellcomprises attenuated expression of the metD gene.
 33. The process ofclaim 32, wherein said bacterial cell is a Coryneform bacterium.
 34. Theprocess of claim 33, wherein the bacterial cell is a Coryneformbacterium from the group consisting of Corynebacterium glutamicum,Corynebacteriurium acetoglutamicum, Corynebacterium acetoacidophilum,Corynebacterium melassecola, Corynebacterium thermoaminogenes,Brevibacterium flavum, Brevibacterium lactofermentum, and Brevibacteriumdivaricatum.
 35. The process of claim 32, wherein the metD genecomprises the polynucleotide sequence of SEQ ID NO.
 1. 36. The processof claim 32, wherein the L-amino acid is L-methionine.
 37. The processof claim 32, wherein the bacteria further comprises at least one genewhose expression is enhanced, wherein the gene is selected from thegroup consisting of the gap, tpi, pgk, zwf, pyc, lysC, hom, metA, metB,aecD, metY, and glyA.
 38. The process of claim 32, wherein the bacteriafurther comprises at least one gene whose expression is attenuated,wherein the gene is selected from the group consisting of the pck, pgi,poxB, thrB, thrC, metK, and ddh.
 39. The process of claim 32, whereinthe bacterial cell is Corynebacterium glutamicum strainATCC13032deltametD.
 40. An isolated polypeptide comprising the sequenceof SEQ ID NO.
 2. 41. An isolated polypeptide comprising an amino acidsequence, which is at least 70% identical to the peptide of claim 40.42. A process for the preparation of an animal food additive, comprisinga) culturing at least one L-methionine-producing microorganism in afermentation medium; b) removing water from the fermentation medium; c)removing from 0 to 100 wt. % of the biomass from the fermentation mediumformed during the culturing; and d) drying the fermentation medium. 43.The process according to claim 42, wherein the at least onemicroorganism comprises genes of the biosynthesis pathway ofL-methionine are that are enhanced.
 44. The process according to claim42, wherein the at least one microorganism comprises genes of thebiosynthesis pathway of L-methionine are that are attenuated.
 45. Theprocess according to claim 42, wherein the at least one microorganismcomprises a polynucleotide which encodes an metD gene, wherein the metDgene is attenuated.
 46. The process according to claim 42, wherein theat least one microorganism is Corynebacterium glutamicum.
 47. Theprocess according to claim 42, wherein the at least one microorganism isCorynebacterium glutamicum strain ATCC 13032deltametD.
 48. The processaccording to claim 42, further comprising at least one of the followingsteps: e) adding at least one organic substance selected from the groupconsisting of L-methionine and D-methionine to the fermentation medium;f) adding at least one auxiliary substance selected from the groupconsisting of silicas, silicates, stearates, grits, and bran to thefermentation medium; or g) converting the fermentation medium obtainedfrom at least one step selected from the group consisting a), b), c),d), e), and f) into an animal food additive.
 49. The process accordingto claim 48, wherein the converting is performed by coating thefermentation medium with at least one film-forming agent.
 50. An animalfood additive made by the process according to claim 49, wherein theanimal food additive is stable in the stomach of an animal.
 51. Ananimal food additive made by the process according to claim 49, whereinthe animal food additive is stable in the rumen of an animal.
 52. Ananimal food additive made by the process according to claim 49, whereinthe at least one film-forming agent is selected from the groupconsisting of metal carbonates, silicas, silicates, alginates,stearates, starches, gums, and cellulose ethers.
 53. An animal foodadditive made by the process according to claim 42, wherein the foodadditive comprises at most 5 wt % of water.
 54. An animal food additivemade by the process according to claim 42, wherein the food additivecomprises at most 2 wt % of water.
 55. An animal food additive made bythe process according to claim 42, wherein the food additive comprisesfrom 1 wt. % to 80 wt. % L-methionine, D-methionine, D,L methionine, ora mixture thereof based on the dry weight of the animal feedstuffsadditive; and from 1 to 40 wt. % L-lysine, D-lysine, D,L-lysine, or amixture thereof based on the dry weight of the animal feedstuffsadditive.